o4"«
vvEPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory ' i
Research Triangle Park NC 27711
Research and Development
EPA-600/S2-82-007 Jan. 1983
Project Summary
Compilation of Ames
Salmonella typhimurium Plate
Incorporation Test Protocols
Stephanie Toney and Larry D. Claxton
This compilation is meant to serve as
1) a reference for workers in the genetic
toxicology field, 2) a starting point for
creation of reference protocols for those
who need precise Ames test protocols,
and 3) a guide to understanding varia-
tions in test results. The full report, the
result of an informal survey conducted
by the U.S. Environmental Protection
Agency, included laboratories that use
the Ames test routinely. It is a simple
compilation of the submitted protocols
and cover letters. Any information not
pertaining directly to the Ames test has
been omitted and so noted on the cover
sheet for each protocol.
The full report is not intended to make
a comment on the competence or per-
formance of any laboratory. It is impor-
tant to remember that some laboratories
may have Standard Operating Pro-
cedures that are more detailed than sub-
mitted protocols. Some laboratories use
the protocol by Ames et al. as the main
procedure and have sent only their
modifications. However, the compilers
do feel that this report is a useful
reference. It can be a guide for writing
better protocols, and it can provide a
better understanding of the variations of
results from different laboratories. A
companion publication will also be
available. It is a tabulated qualitative sum-
mary and comparison of submitted pro-
tocols.
This Project Summary was developed
by EPA's Health Effects Research Labor-
atory, Research Triangle Park, NC, to an-
nounce key findings of the research pro-
ject that is fully documented in a separate
report of the same title (see Project Re-
port ordering Information at back).
Introduction
Bacterial tests for mutagenicity have
been available for approximately 30
years. In 1951, Demerec et al. found
that 19 out of 31 chemicals tested using
an Escherichia coli reverse mutation
system were mutagenic. After modify-
ing the mutation system described by
Demerec, Szybalski (1958) tested 431
substances for mutagenicity. Recogniz-
ing the inadequacies of the streptomycin
resistance system, Ames (1971) pub-
lished the methods for a bacterial sys-
tem using a histidine-requiring mutant of
Salmonella typhimurium. Mailing (1971)
was the first to merge a mammalian me-
tabolizing system with a bacterial system
in order to demonstrate the mutagenicity
of dimethylnitrosamine.
In 1975, Ames et al. published a de-
tailed protocol that incorporated the use
of a mammalian metabolizing system
thereby establishing the "Ames test" as
a routine screening system for mutage-
nicity and potential carcinogenicity.
Although the 1975 paper by Ames etal.
provided a highly detailed protocol,
many laboratories have introduced a
variety of changes or additions. Also,
summaries of international meetings
have been published (Mattern and Greim,
1977; Seller et al., 1979; de Serres and
Shelby, 1979a; de Serres and Shelby,
1979b; Stich and San, 1979).
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Since so many researchers and meet-
ings have published suggested alterations
and additions to the original protocol, the
U.S. Environmental Protection Agency
(EPA) decided to conduct an informal
survey of laboratories that use the Ames
test. A list of laboratories performing the
Ames test in a routine manner was ob-
tained from Dr. Mike Shelby at the Na-
tional Institute of Environmental Health
Sciences, Research Triangle Park, NC.
An initial letter was sent to 46 labora-
tories included in this list. A follow-up
letter was sent to the laboratories who
had not responded within a one-month
interval. As a result 33 laboratories
responded to the request. The responses
included 6 laboratories that no longer
performed the Ames test, 1 that had
closed, 23 that did perform the test and
submitted protocols, and 3 that per-
formed the test but elected to restrict
the usage of their protocols. In addition,
two EPA laboratory protocols are in-
cluded within this compilation. A list of
names and addresses of the laboratories
that participated by sending protocols is
found in Table 1.
Table 1. Names and Addresses of
Participating Laboratories
Dr. Andrew Sivak
Arthur D. Little, Inc.
25 Acorn Park
Cambridge, MA 02140
Dr. James P. Crowley
Battelle Columbus Laboratories
505 King Ave.
Columbus, OH 43201
Dr. Douglas M. Hanson
Bioassay Systems Corporation
225 WildwoodAve.
Woburn, MA 01801
Dr. Clyde R. Goodheart
Biolabs, Inc.
2910MacArthurBlvd.
Northbrook, IL 60062
Dr. Rae E. Drazin
Bio-Technics Laboratories, Inc.
1133 Crenshaw Blvd.
Los Angeles, CA 9O019
Dr. William T. Speck
Case Western Reserve University
Department of Pediatrics
2103AdelbertRd.
Cleveland, OH 44106
Dr. Steve R. Haworth
EG&G Mason Research Institute
1530 E. Jefferson St.
Rockville, MD 20852
Dr. Larry D. Claxton
Genetic Toxicology Division
U.S. Environmental Protection Agency
Research Triangle Park, NC27711
Dr. Vincent F. Simmon
Genex Corporation
6110 Executive Blvd.
Suite 1090
Rockville, MD 20852
Ms. Nancy E. McCarroll
Hazleton Laboratories America, Inc.
9200 Leesburg Tnpk.
Vienna, VA 22180
Dr. George C. Lavelle
Hill Top Research, Inc.
Department of Toxicology
Miamiville, OH45147
Mr. Peter W. Barbera
IIT Research Institute
Life Sciences Division
10 West 35th St.
Chicago, IL 60616
Ms. Shirley Louie
Jefferson Professional Services
P.O. Box 3397
Little Rock, AR 72203
Dr. Andrew M. Tometsko
Litron Laboratories, Ltd.
1351 Mount Hope A ve.
Rochester, NY 14620
Dr. David Brusick
Litton Bionetics, Inc.
5516 Nicholson Ln.
Kensington, MD 20795
Dr. Carol L. Richardson
Meloy Laboratories, Inc.
6715 Electronic Dr.
Springfield, VA 22151
Mr. Andrew M. Losikoff
Microbiological Associates
5221 River Rd.
Bethesda,MD20016
Ms. JoAnne Gridley
Monsanto Research Corp.
1515 Nicholas Rd.
Dayton Laboratory
Dayton, OH45407
Dr. John E. Preston
U.S.E.P.A. - NEIC
Bldg. 53
P.O. Box25227
Denver, CO 80225
Dr. Bruce C. Casto
Northrop Services, Inc.
P.O.Box 12313
Research Triangle Park, NC 27709
Dr. Robert A. Finch
Raltech Scientific Services
A Division of Ralston Purina Co.
P.O. Box 7545
Madison, Wl S3 70 7
Mr. Thomas J. Hughes
Research Triangle Institute
P.O.Box 12194
Research Triangle Park, NC 27709
Dr. Nathan D. Greene
Southwest Foundation for Research
and Education
P.O. Box 28147
San Antonio, TX 78284
Dr. David C.L. Jones
SRI International
333 Ravenswood Ave.
Menlo Park, CA 94025
Ms. Melanie Baltezore
ES Unilab Research, Inc.
2800 Seventh St.
Berkeley, C A 94710
This publication is a simple compilation
of the protocols and cover letters sub-
mitted. Any submitted information not
pertaining directly to the Ames Salmo-
nella typhimurium plate incorporation
test have been omitted; however, any
omissions are indicated at the beginning
of each protocol. The compilers and EPA
understand that these protocols may not
accurately reflect the competence or
performance of any laboratory. Some
laboratories may maintain Standard
Operating Procedures (SOPs) that are
more detailed than the submitted proto-
cols, and some may have modified or ex-
panded their procedures since submis-
sion of this document. However, the
compilers do feel that many will find this
report a useful reference, a guide for
writing better protocols, and a guide to
understanding why variations in results
from various laboratories exist. A com-
panion volume will be available which
tabulates and discusses the similarities
and differences between the submitted
protocols.
Description of the Ames
Salmonella typhimurium Plate
Incorporation Test
The .Ames test is a short-term bioassay
for mutagenicity testing. The advantages
of this test are the speed with which re-
sults can be obtained, usually three to
four days, and the relative low cost. The
Ames test is used for testing both pure
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hemicals (cyclophosphamide and 6-
lercaptopurine) and complex mixtures
icluding mobile source emissions (diesel
nd gasoline) and comparative source
missions (coke oven, roofing tar, and
igarette smoke condensate).
The Ames test basically involves taking
given sample and adding it to a strain of
'almonella in an agar overlay tube that is
ten plated on minimal media (see Figure
). The sample is routinely tested at sev-
ral dose levels with five Ames strains of
'almonella, with and without metabolic
ctivation. The dose levels are plated
sually in duplicate or triplicate. This
lating involves approximately 120
lates/sample excluding the control
lates.
After the bacteria, test compound,
nd possibly metabolic activation are
dded to the overlay tube, the contents
re gently mixed and plated out on
'ogel-Bonner minimal media plates.
hese plates are incubated for three
ays at 37 °C in the dark. After three
ays the plates are scored for mutants.
Five Ames strains are in widespread
se—TA1 537, TA1 538 and its deriva-
ve TA98, and TA1 535 and its deriva-
ve TA100. These strains are all histi-
ne-dependent mutants and revert to
/ild type in the Ames test. Two of the
trains detect frameshift mutations
FA1 537 and TA1538), and one strain
etects base-pair substitutions (TA1535).
itrainsTA98 andTA100are less specif-
c as to the type of mutants they detect.
trains TA98 and TA100 both contain
n R factor plasmid that increases the
ensitivity of these strains. All five
trains have a rfa mutation creating a
eficiency in the lipopolysaccharide cell
vail therefore increasing permeability to
nacromolecules. The strains all have a
ivr B mutation that decreases genetic
eparability .
The mammalian metabolic activation
ystem (S-9) is added to the overlay to
dentify compounds that require the
netabolic activation mechanisms of
nammals not found in bacteria. The
nammalian metabolic activation system
icrmits metabolites of the test com-
lound to be tested for mutagenicity.
kmes' activation system generally con-
iists of a 9000 x g supernatant of
kroclor-1254-induced rat liver homoge-
late.
Bacteria
S-9 microsomes
\
Chemical
T V
tt
Incubate
48 hours, 37°C
Soft agar overlay
Plate with
overlay
Results:
Control
Increasing dosage
(Mutagenic with no killing)
Figure /. Schematic for plate incorporation test.
Stephanie Toney is with Northrop Services, Research Triangle Park, NC 27709;
the EPA author Larry D. Claxton (also the EPA Project Officer, see below) is
with the Health Effects Research Laboratory, Research Triangle Park, NC
27711.
The complete report, entitled "Compilation of Ames Salmonella typhimurium
Plate Incorporation Test Protocols," (Order No. PB 83-113 290; Cost: $37.00,
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
•h U.S. GOVERNMENT PRINTING OFFICE: 1983 659-OI7/O88S
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